1. Field of the Invention
The present invention relates to the biological conversion of cellulosic substrates into fuels and chemicals, and in particular to recombinant Zymomonas mobilis strains which ferment xylose, arabinose, and mannose or all of these into ethanol.
2. Description of the Related Art
Fermentation technology is useful for the conversion renewable biomass cellulose substrates into fuels and chemicals, such as ethanol. A typical substrate is comprised of 35–45% cellulose, 30–40% hemicellulose, and 15% lignin. The hydrolysis fraction contains glucose polymers, and the hemicellulose fraction contains mostly xylose. Arabinose is also a significant fermentable substrate found in biomass materials, such as switchgrass grass and corn fiber. Thus, achieving a high rate of specific product formation and conversion efficiency in the fermentation of the pentose sugars is vital to the commercial production of fuels and chemicals from a renewable substrates.
Z. mobilis is widely reported for its ability to rapidly and efficiently convert glucose substrates into ethanol, at a low pH, in an anaerobic culture, and in a medium which contains the inhibitory compounds typically associated with a lignocellulose-hydrolysate. A distinct disadvantage in the use of Z. mobilis is, however, that it does not ferment pentose sugars. To overcome this disadvantage, the prior art has focused on recombinant Z. mobilis strains which ferment a mixture of glucose, and xylose or arabinose, or both, using exogenous genes which catalyze the metabolism of xylose and arabinose. These strains, and the cloning vectors, are based on the use of multiple-copy plasmids having antibiotic resistance markers.
U.S. Pat. No. 5,514,583 relates to a transformed Z. mobilis xylose fermenting strain (CP4/pZB4 and pZB5) having exogenous genes, and plasmid vectors (pZB4 and pZB5) encoding xylose isomerase, xylulokinase, transaldolase and transketolase, and further comprising at least one promoter (Pgap and Peno) recognized by Zymomonas which regulates the expression of at least one of said genes. The microorganism is capable of growing on xylose as a sole carbon source, and fermenting xylose to ethanol at about 88% of the maximum theoretic yield. U.S. Pat. Nos. 5,712,133 and 5,726,053 relates to, inter alia, Z. mobilis arabinose fermenting transformants (39676/pZB 206), containing exogenous genes that encode L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate-4-epimerase, transaldolase and transketolase which impart arabinose to ethanol fermentation capability. The plasmid vector (pZB 206) and a process of using the transformants of the fermentation of a glucose and arabinose containing substrate is also described. U.S. Pat. No. 5,843,760 discloses a Z. mobilis xylose and arabinose fermenting transformant (206C/pZB301) containing exogenous genes encoding xylose isomerase, xylulokinase, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-phosphate 4-epimerase, transaldolase and transketolase, and further comprising at least one promoter recognized by Zymomonas which regulates the expression of at least one of said genes, wherein said microorganism is capable of growing on arabinose and/or xylose, alone or in combination, as the carbon source and fermenting said arabinose and xylose to ethanol. The process of using the transformants together with the plasmid vectors (pZB301, pZB401, pZB402, and pZB 403) is also described. Although hybrid plasmids may be readily maintained in Z. mobilis when cultivated in a monoculture under controlled conditions, they frequently become unstable when the host organism is grown in the absence of selection pressure for plasmid maintenance, i.e., in the presence of antibiotics. For example, the exogenous genes in the above referenced strains are capable of stable expression for about forty generations. Instability may be exacerbated when Z. mobilis has to compete with other organisms in a mixed culture, such as a cellulose simultaneous-saccharification-fermentation process. In addition, antibiotic resistance markers are generally undesirable for industrial application, such as the large-scale production of ethanol. Thus, it is preferable to insert the cloned genes into the Z. mobilis genome, where they are maintained at a low, natural copy number, and are thus not over-expressed, and where, they should be as stable as genomic DNA.
In Escherichia coli, the classical method for generating genomic inserts of foreign genes involves the use of specialized 1 phage cloning vectors that can exist stable in the lysogenic state. Alternatively, genes can be inserted through homologous recombination, when bracketed with E. coli chromosomal sequences, or by transposition if the genes can be cloned in the permissive sites of a transposon. While transposition has been demonstrated in Z. mobilis, (Pappas, K. M., et al., (1997) Journal of Applied Microbiology, 82: 379–388), it has been limited to mini Mm or Tn5 multiple transposition of random auxotrophy or antibiotic resistance phenotypes for genetic analysis. In the case of the Tn5 derivatives the insertion is reportedly stable for only 5–15 generations (Pappas, K. M., et seq. P. 383, FIG. 1.) Moreover, site-specific insertion through homologous recombination in Z. mobilis was not demonstrated, and no bacteriophage has ever been isolated from Zymomonas. 
Transposons Tn5 and Tn10 are well known and have been widely used for mutagenesis and insertion of cloned DNA into a variety of gram-negative bacteria. In Herrero, M., et al., (1990) (J. Bacteriol. 172:6557–6567), a procedure is described for cloning and stable insertion of foreign genes into the chromosome of gram-negative eubacteria by combining two sets of plasmids, (i) the transposition features of Tn10 and Tn5, (ii) the resistance to certain compounds, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contain unique NotI or SfiI, sites internal to either the Tn10 or the Tn5 inverted repeats. These sites are used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions are maintained only in donor host strains that produce the R6K-specified p protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons are transformed into a specialized lpri lysogenic E. coli strain, such as E. coli Sm10(lpir), with a chromosomally integrated RP4 that provided broad-host range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria is accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target is mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function is not maintained in the target cells, such cells are immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers.
Herrero, M. et al., (1990), (Journal of Bacteriol. 172(11): 6568–6572), relates to the construction of a collection of Tn5-derived minitransposons, such as Tn5Tc. It may be possible to employ the Tn5-derived minitransposons, such as Tn5Tc, to incorporate foreign DNA fragments into the genome of a variety of gram-negative bacteria. The minitransposons consist of genes specifying the resistance to kanamycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Therefore, insertions produced by these elements are generally more stable because of the absence of transposase-mediated secondary transpositions, deletions, and inversions. See also, Berg et al., (1989) Transposable elements and the genetic engineering of bacteria, p.p. 879–926, in D. E. Berg, Mobile DNA, American Society of Microbiology, Washington, D.C. Stable insertions can in this way be obtained with elements derived, for instance also from Tn10. Way, J. C. et al., (1984) (Gene 32: 369–379).
The structure of mini-Tn5Tc, Herrero, et seq., p. 6569, is described for use for insertion mutagenesis or as a transposon vector for the cloning of DNA fragments flanked by NotI sites (isolated by cloning DNA fragments first into the pUC18 derivatives pUC18Not and pUC18Not). The Mini-Tn5Tc element is constructed, in vitro, using standard recombinant DNA techniques. Maniatis, T., et al., (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The determinant for tetracycline (Tc) resistance is obtained as an EcoRI fragment from plasmids bearing them as an interposon. Fellay, R., et al. (1987) Interposon mutagensesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertion mutagenesis of Gram-negative bacteria. Gene 52: 147–154. The fragment is subsequently cloned into a single EcoRI site of pUC18Sfi, excised as an SfiI fragment, and inserted between the Tn5 19-base pair termini in pUT so that the mobile unit is present in all cases as an XbaI-EcoRI (partial) portion of the delivery plasmid. The resulting element is mini-Tn5Tc.
Tn10-based transposon vector delivery systems are described generally in Herrero, M. et seq. 172:6557–6567. Phage 1, a derivative IRP167, carries a 5.1-kb EcoRI insert containing the mini-Tn10 Km element and the transposase gene of IS10R is located outside the inverted repeats of the mobile element and downstream of the PTac promoter. To obtain a transposon delivery plasmid with a host-independent regulation of its transposition, the EcoRI insert fragment is ligated to pBOR8, a derivative of pGP704 containing laclq gene from plasmid pMJR1560. This plasmid is unable to replicate in host strains devoid of the R6K-specified p protein product of the pir gene. pGP704 contains the conjugal transfer origin (oriT sequence) of the RP4 plasmid and can therefore be transferred to gram-negative bacterial when provided in trans with mobilization (Mob) functions. The MluI fragment internal to the inverted repeats containing the original-specified p protein product of the original kanamycin resistance gene of the mini-Tn10 is replaced by a fragment containing a SfiI-Ptt cassette, appropriately modified by the addition of the NotI site and the MluI adapters, which produced the pLODPtt. This construction has unique SfiI, NotI, and XbaI cloning sites between the mini-Tn10 inverted repeats. The Ptt resistance marker (Ptt′) of pLOFPtt is exchanged by kanamycin resistance to produce plasmid pLOFKm.
Difficulty continues to exist in the area in genetic manipulation of Zymomonas mobilis to produce genetically stable strains that contain the genetic components necessary for pentose sugar fermentation in ethanol production. The genetic components necessary to permit Zymomonas to utilize pentose sugars would encode xylose isomerase, xylulokinase, transaldolase and transketolase.
In view of the foregoing, a need exists for the construction of stable recombinant Z. mobilis strains which are capable of fermenting pentose sugars, such as xylose and arabinose, or both, to ethanol. A need therefore exists for the generation of stable genomic inserts that encode the enzymes necessary for pentose sugar catabolism. A need continues to exist for commercially suitable strains of such Zymomonas, which means that such strains should also be free of antibiotic resistance, and stable for at least 40 generations or more in non-selection media. Commercially valuable strains must also preferably demonstrate a high specific rate of product formation at close to maximum theoretical product yield. These and other deficiencies in the art of microbial ethanol production are addressed with the present invention.